In particular, conventional cryoprotectants were added to PLGA nanoparticle and liposome formulations in various conditions. Additionally, hyaluronic acid (HA), known for its ability to target the CD44 receptor, was assessed as a cryoprotective excipient: it was added to the nanocarriers as either a free molecule or conjugated to a phospholipid to increase the interaction with the polymer or lipid matrix while exposing HA on the nanocarrier surface. The formulations were resuspended and characterized for size, polydispersity index, zeta potential and morphology. It was demonstrated that only the highest percentages of cryoprotectants allowed the resuspension of stable nanocarriers. Moreover, unlike free HA, HA-phospholipid conjugates were able to maintain the particle mean size after the reconstitution of lyophilized nanoparticles and liposomes. This study paves the way for the use of HA-phospholipids to achieve, at the same time, nanocarrier cryoprotection and active targeting.Biodegradable nanocarriers represent promising tools for controlled drug delivery. However, one major drawback related to their use is the long-term stability, which is largely influenced by the presence of water in the formulations, so to solve this problem, freeze-drying with cryoprotectants has been proposed. In the present study, the influence of the freeze-drying procedure on the storage stability of poly(lactide- Applications of nanoparticulate systems for drug delivery have been extensively discussed over the years for their promising therapeutic and diagnostic purposes ,2,3. IndFreeze drying, also named lyophilization, is the most used method to remove water and handle the long-lasting stability of nanoparticle formulations ,10. As aSince they show the ability to act as integrity membrane protectants, short-chain saccharides are the preferable aid-molecules useful to minimize nanoparticle instability during the freezing steps of lipid and polymer nanoparticles ,21,22. Tco-glycolide) (PLGA) nanoparticles and liposomes at different concentrations. Moreover, a comparative study with hyaluronic acid (HA) at a low molecular weight (4.8 kDa and 14.8 kDa) as a free saccharide or conjugated to a phospholipid [Besides the reported studies on conventional cryo- and lyoprotectants, to the best of our knowledge, a comparative study between polymer nanoparticles and liposomes stabilized with short and long-chain saccharides has not been assessed yet. To this aim, we selected sucrose, trehalose, mannitol and glucose as cryoprotectants and tested their protective activity on poly, PLGA 75:25 , PEG2000-PLGA 50:50 (PLGA 11.5 kDa), D-(+)-glucose, D-(+)-sucrose, D-(+)-trehalose, D-(+)-mannitol (purity minimum 95%) and solvents  were purchased from Merck . Sodium hyaluronate (HA) (4.8 kDa or 14.8 kDa) was purchased from Lifecore Biomedical . The compound 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was conjugated to 4.8 kDa or 14.8 kDa HA (HA4.8-DPPE or HA14.8-DPPE) using the method described by Arpicco et al. [® water  was used. Solvents were evaporated using a rotating evaporator  equipped with a vacuum pump (Diaphragm Vacuum Pump DC-4).All the phospholipids, cholesterol (CHOL), PLGA 50:50 . Further batches were prepared by 2:1 (w/w) polymer blends between PLGA (50:50 or 75:25) and PEG2000-PLGA 50:50, as previously described  [PLGA nanoparticles were prepared by the nanoprecipitation technique . To this6 mg/mL) .sn-glycero-3-phosphocholine (DPPC), CHOL and L-α-phosphatidylglycerol (PG) in 70:30:3 molar ratio. Lipids were dissolved in chloroform and then evaporated by a rotary evaporator. The resulting thin film was dried under vacuum overnight and then hydrated with 900 µL of 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES) buffer 0.02 M pH 7.4, vortexed and bath sonicated. Afterward, the obtained suspension was extruded  at 60 °C under nitrogen through a 200 nm-polycarbonate membrane .Liposomes were prepared using the thin lipid film-hydration method by mixing 1,2-dipalmitoyl-All the suspensions were stored at 4 °C until further use.4.8-DPPE or HA14.8-DPPE conjugates were added in the aqueous phase before the addition of the polymer acetone solution during the nanoprecipitation procedure. Practically, 3.1 mg of HA4.8-DPPE or 15.3 mg of HA14.8-DPPE were added to 1 mL of MilliQ® water. Then, for each preparation, 6 mg of PLGA (50:50 or 75:25) were dissolved in 500 µL of acetone and poured into the aqueous phase containing the HA derivative under magnetic stirring. Acetone was then eliminated under rotary evaporation.For PLGA nanoparticles, HA4.8-DPPE or HA14.8-DPPE conjugates (3 molar ratio) in 900 μL of HEPES buffer during the hydration of the lipid film.HA-decorated liposomes were prepared as described above for plain liposomes by adding to the phospholipid mixture (composed of DPPC/CHOL 70:30 molar ratio) HAOnce the development of nanosystems was optimized, PLGA nanoparticles and liposomes (both blank and HA-decorated) were freeze-dried with an Alpha 1–4 LSCplus freeze-drier . In addition, different saccharides  were evaluated as cryoprotective agents for blank nanoparticles or liposomes.w/w) either in the aqueous phase prior to the addition of acetone to the aqueous solution or after the evaporation of the organic solvent. Moreover, the addition of free HA was compared with the insertion of HA-DPPE conjugates: to this aim, free 4.8 kDa HA (1.31 mg) or free 14.8 kDa HA (3.95 mg) were added to nanoparticle suspensions after organic solvent evaporation.In particular, for PLGA nanoparticles, mannitol, glucose, sucrose or trehalose were added at the concentration of 2.5, 5, 10 or 20% (w/w) sugar:lipids ratio or only to the resulting liposome suspensions. Free HA was added in the same conditions in a 3% molar ratio.Sucrose, trehalose and free HA were selected to test the cryoprotective effect on liposomes. Sucrose and trehalose were added directly both to the hydration buffer and after liposome formation in a 5:1 . Finally, a secondary drying step was carried out at 25 °C and 0.010 mbar for 3 h.® water or HEPES buffer as before freeze-drying.To test the cryoprotective effect, freeze-dried formulations were then resuspended by vortex-mixing (and bath sonication for PLGA nanoparticles) in the same volume of MilliQ® water. Each measurement was performed in triplicate both before and after the freeze-drying process. The particle surface charge of the formulations was investigated by zeta potential measurements at 25 °C using the Smoluchowski equation and the Nanosizer Nano Z after 1/10 dilution of the suspensions in MilliQ® water. Each value reported is the average of three measurements both before and after the freeze-drying process.The mean particle hydrodynamic diameter and the polydispersity index (PDI) of the different nanoparticle and liposome samples were determined at 25 °C by quasi-elastic light scattering (QELS) using a nanosizer . The selected angle was 173°, and the measurement was made after 1/10 dilution of the particulate suspensions in MilliQFinally, the physical stability of resuspended nanoparticles and liposomes was determined in the storage conditions (4 °C) by evaluating mean hydrodynamic diameter, PDI and zeta potential at different interval times during 4 weeks at 4 °C .4.8-DPPE-decorated nanoparticles and liposomes were examined both before and after the freeze-drying process as well as after further resuspension. For analyses, one drop of the as prepared and resuspended samples was placed on an aluminum stub coated with a conducting adhesive and left to dry in air at room temperature. As for the freeze-dried samples, the powder was briefly contacted with the same kind of stub described previously. All samples were then simply submitted to metallization with Cr (ca. 5 nm) to avoid any charging effect  and inserted in the chamber by a fully motorized procedure.In order to have information on the morphology and size of the HA-decorated nanoparticles and liposomes, Field Emission Scanning Electron Microscopy (FESEM) analyses were performed by a Tescan S9000G FESEM 3010 microscope  working at 30 kV, equipped with a high brightness Schottky emitter and fitted with Energy Dispersive X-ray Spectroscopy (EDS) analysis by an Ultim Max Silicon Drift Detector . HAFreeze drying allows drug delivery systems, such as nanoparticles and liposomes, to be obtained and stored as dry forms, thus avoiding chemical and physical instability and improving long-term storage ,34. Howew/w) to PLGA or PEG2000-PLGA nanoparticle samples according to the percentages reported in the literature [Glucose, sucrose, trehalose and mannitol were added between 2.5% and 20%  and PEGThe zeta potential values measured for all PLGA nanoparticles were found in the range between −36 mV and −21 mV, regardless of the nanoparticle composition; these values allow nanoparticles to be stable thanks to charge repulsion.® water. All the samples could be resuspended except for nanoparticles without cryoprotectant and those with mannitol, which gave a precipitate. After lyophilization, these samples appeared as a very compact powder, which probably hampered the rehydration. On the contrary, the formulations with glucose, sucrose or trehalose were able to be resuspended. Nevertheless, only the highest percentages of sucrose and trehalose .w/w) was used since it was reported to be able to maximize the stabilization effect, in particular in the case of disaccharides [The effect of disaccharides as protective agents in the freeze-drying process was also assessed on liposomes. Liposomal formulations were prepared by hydration of the lipid film followed by extrusion through polycarbonate filters to obtain homogenous small unilamellar vesicles. Sucrose and trehalose were investigated for their ability to prevent aggregation of liposomes after freeze-drying  since dicharides . On the charides .Thus, to set a comparative study between polymer and lipid nanoparticles, further tests were performed by adding the cryoprotectants after nanosystem formation (“out” addition method). As a first result, we confirmed that, as for polymer nanoparticles, for lipid-based nanocarriers, significant amounts of cryoprotectants are required to supersaturate the surface and lead to the formation of a glassy matrix all around their structure for efficient carrier protection during lyophilization .After considering different sugars, we moved towards the use of a polysaccharide, namely HA, which is an anionic glycosaminoglycan already studied for the cryopreservation of cell membranes . FurtherFree HA (4.8 kDa or 14.8 kDa) was directly added to the final PLGA (75:25 or 50:50) nanosuspensions, and then the freeze-drying procedure was carried out. After the addition of water, lyophilized samples could not be resuspended, as they formed large aggregates. We can thus highlight that free HA at the two considered molecular weights does not have a protective effect on PLGA nanoparticles during the freeze-drying process.The potential cryoprotectant effect of free HA was also assessed on liposomes. As for nanoparticles, HA was added directly to the final liposomal suspensions. Characterization of liposomes after freeze-drying in the presence of free HA showed a weak cryoprotectant effect for HA 14.8 kDa. On the contrary, a significant size increase was observed for HA at a lower molecular weight .4.8-DPPE) by the freeze-drying process, thanks to the presence of HA-DPPE in the formulations. Comparable results were achieved for liposomes, which showed a size variation below 20% (4.8-DPPE or HA14.8-DPPE).In the further set of experiments, we evaluated the effect of HA as a cryoprotectant agent when it is associated with polymer- and lipid-nanocarrier structure by hydrophobic interactions. Since it is highly hydrophilic, HA (4.8 kDa or 14.8 kDa) was linked to the DPPE phospholipid, which is able to associate with a polymer matrix or phospholipid bilayer and obtain HA-coated nanoparticles. These conjugates have already been used in several settings to prepare HA-decorated liposomes for cell targeting that have been proposed for either intravenous or cutaneous administration ,48,49,50elow 20% . All theConcerning zeta potential values, the addition of HA-DPPE conjugates lowered the surface charge of the nanocarriers (between −53 mV and −40 mV for PLGA nanoparticles and between −43 mV and −31 mV for liposomes) due to the presence of negatively charged functional groups of HA. After lyophilization, the overall obtained resuspended formulations maintained the strongly negative zeta potential, thus guaranteeing colloidal stability through electrostatic repulsion forces, according to the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory .4.8-DPPE/PLGA 75:25 nanoparticles, and HA4.8-DPPE-liposomes further validated the beneficial effect of HA-DPPE in the formulations , whereas HA4.8-DPPE-liposomes displayed a slightly globular shape (d) due to the drying step carried out to prepare the sample for the measurements . As a consequence, the process conditions have to be selected by testing different experimental settings. Moreover, this type of study has to be repeated for each modification in the nanocarrier composition, e.g., the addition of an active principle or a fluorescent dye. Concerning our results, only the highest percentages (10–20%) of sucrose and trehalose showed to be able to act as cryoprotectant agents for both PLGA nanoparticles and liposomes.Besides the classical saccharides used during the freeze-drying process, this work proposes the use of HA of different molecular weights as either a free molecule or conjugated to a phospholipid to achieve nanocarrier lyophilization. Although a moderate effect was observed for free HA-associated liposomes, more interesting are the results with HA-DPPE conjugates for both PLGA nanoparticles and liposomes: thanks to the lipophilic moiety, these molecules showed to be able to anchor to the PLGA matrix or to the liposome bilayer and to stably associate HA to the nanocarrier surface; in this way, HA-DPPE-decorated nanoparticles and liposomes can be resuspended after freeze-drying. This approach is particularly interesting since the HA moiety could act not only as a cryoprotectant agent but also as a targeting molecule towards the CD44 receptor, which is highly expressed in several cancer cells. Future steps will concern the evaluation of the ability of resuspended HA-associated nanoparticles and liposomes to target the CD44 receptor.


Background: Microsporidia are a group of obligated intracellular fungus pathogens. Monocytes and the derivative macrophages are among the most important players in host immunity. The invasion of microsporidia may significantly affect the monocytes maturation and extravasation processes. Methods: We utilized a previously established microsporidia infection murine model to investigate the influences of microsporidia Encephalitozoon hellem (E. hellem) infection on monocyte maturation, releasing into the circulation and extravasation to the inflammation site. Flow cytometry and qPCR analysis were used to compare the monocytes and derivative macrophages isolated from bone marrow, peripheral blood and tissues of E. hellem-infected and control mice. Results: The results showed that the pro-inflammatory group of CD11b+Ly-6C+ monocytes are promoted in E. hellem-infected mice. Interestingly, the percentage of Ly-6C+ monocytes from E. hellem-infected mice are significantly lower in peripheral blood while significantly higher in the inflamed small intestine, together with up-regulated ratio of F4/80 macrophage in small intestine as well. Conclusions: Our findings demonstrated that E. hellem infection leads to promoted monocytes maturation in bone marrow, up-regulation of extravasation from peripheral blood to inflammation site and maturation into macrophages. Our study is the first systematic analysis of monocytes maturation and trafficking during microsporidia infection, and will provide better understanding of the pathogen–host interactions. Encephalitozoon hellem belongs to microsporidia which is defined as a huge group of eukaryotic, intracellular pathogens. They can infect a variety of hosts including human, specifically the immunocompromised populations [ulations . The comulations ,3,4. Sinulations , the resNow we know that the host innate immunity plays a significant role against microsporidia, such as the interactions between microsporidia and macrophages ,7,8. As + is usually considered as specific marker for “inflammatory” monocytes [+ monocytes. Previous research has shown that, at the post-infection stage of Encephalitozoon cuniculi or Encephalitozoon intestinalis, CC-chemokines of CCL2, CCL7, et al., were upregulated [+ monocytes which further traffic from bone marrow to the blood circulation and derive to inflammatory macrophages with high expression of F4/80 [L. monocytogenes, blood monocytes decrease during this time, as reported previously [Monocytes and the derivative macrophages, regarded as the immediate arm of the immune system, have the essential function of effecting and regulating the innate immune response ,12. Monoonocytes ,14,15,16egulated . Both CCof F4/80 . During of F4/80 ,19. For eviously .E. hellem infection affects monocytes maturation and extravasation, we used the established murine model [E. hellem-infected and uninfected control mice, flow cytometry and qPCR analysis were applied. Our study will help us to better understand the long-term influences of microsporidia infection on host immunity establishment.To fill the gap in knowledge of how ne model . MonocytSpecified pathogen-free, 6-week-old female C57BL/6 mice were purchased and reared in animal care facility. All of the experiment mice were conducted following the Southwest University-approved animal protocol (SYXK-2017-0019).Encephalitozoon hellem strain (ATCC 50504/50451), gifted by Prof. Louis Weiss , was used in this study. The spores were reproduced in rabbit kidney cells  supplied with 5% CO2 in Minimum Essential Medium Eagle (MEM) containing 10% fetal bovine serum (FBS)  and penicillin-streptomycin. The spores were collected from culture media and then passed through 5 µm-size filter  to remove host cells, then concentrated by centrifugation and stored in sterile distilled water at 4 °C [ at 4 °C .This study used a previously established mouse infection model, which uses dexamethasone to transiently suppress mice immunity for a short period of time . Dexamet7E. hellem spores per day for two days. Controls were uninfected mice. All the mice sacrificed on the 17th day after infection, and samples of bone marrow (from hind legs), peripheral blood, about 0.32 g of small intestine were collected from each group. TRIzol method was used to extract the total RNA of samples. CTAB method was used for the extraction of the whole genome in tissues. PCR assay was used to prove the infection.The mice were then intraperitoneally injected with 1 × 10Sterile scissors were used to open the abdominal cavity, then removed the surface muscles to find the pelvic-hip joint. A sterile blade and tweezers were used to remove the muscles, adipose tissues and other surrounding tissues. Then, the knee joint was cut off, and the operation was carried out in the plate containing PBS . Both en6 cells were recorded. Through the support of the laboratory, flow cytometry analysis could be performed by Beckman Coulter CytoFlex S, and the data were analyzed by CytExpert Software.The isolated bone marrow single cells were used for the detection of surface marker expressions, CD11b and Ly-6C, by flow cytometry. 200 μL immune cell resuspension was taken out of each sample and filtered by cell strainers. The cell resuspension was then stained for 30 min at 4 °C by APC-conjugated anti-CD11b and PE-conjugated anti-Ly-6C . About 10E. hellem-infected and the uninfected control. Sodium citrate was used as anticoagulant, and blood samples were centrifuged at 400× g for 10 min to enrich plasma. All samples were then subjected to qPCR analysis.Peripheral blood samples were collected from mice in both groups—the E. hellem in the host tissues was proved through PCR assay, using primers 5′-CACCAGGTTGATTCTGCCTGACGT-3′ and 5′-CCTCTCCGGAACCAAACCCTGAT-3′, 279 bp of E. hellem gene fragment can be amplified.The colonization of ® 1st Strand cDNA Synthesis SuperMix for qPCR . qPCR was performed through Roche LightCycler® 96 System  with the use of Hieff® qPCR SYBR® Green Master Mix . Actin was used as the internal reference in the whole investigation. Primers were either synthesized by using the Primer Premier 5.0 program or supported by previous studies. The sequences were shown as follows: By the method of TRIzol, total RNA from mice bone marrow cells, peripheral blood cells and intestines were extracted. Reverse transcription was conducted by using of HifairCD11b, 5′-CAATAGCCAGCCTCAGTGC-3′ (forward) and 5′-GAGCCCAGGGGAGAAGTG-3′ (reverse);Ly-6C, 5′-GCAGTGCTACGAGTGCTATGG-3′ (forward) and 5′-ACTGACGGGTCTTTAGTTTCCTT-3′ (reverse);F4/80, 5′-ACCTAGACATCGAAAGCAAA-3′ (forward) and 5′-TGATTATGAAACAGCCAACA-3′ (reverse);CCR2, 5′-GAGTGAGAAGGAGGAGATATGC-3′ (forward) and 5′-AACACAGATAGGAGAAGGAACC 3′ (reverse);Actin, 5′-GCTGTCCCTGTATGCCTCTG-3′ (forward) and 3′-TGATGTCACGCACGATTTCC-5′ (reverse).t-tests were implemented to figure out the difference. p < 0.5 was deemed to be the indication of significant difference between two groups.For statistical analysis and groups comparison, one-way ANOVA and Student’s E. hellem in the host tissue, a PCR assay was used. The result showed that E. hellem successfully infected the mice  are promoted to get proliferation in the bone marrow under the circumstances of E. hellem infection.We further investigated the expressions of CD11b and Ly-6C on bone marrow cells by flow cytometry analysis. We firstly assigned the monocytes as low granule content and high CD11b-expression cells among all bone marrow cells A. The excontrols B,C. ThesE. hellem-infected and uninfected groups. qPCR was applied for the detection of the expression level of CD11b and Ly-6C in murine peripheral blood. The result indicated that although the expression of general marker of monocytes and other leukocytes showed significant upregulation  is necessary for monocytes egression from bone marrow and recruitment from the blood to inflamed tissue. The expression of CCR2 was detected in bone marrow, peripheral blood and small intestine through relative quantification. We found that the expression level of CCR2 shows upregulation in bone marrow A, but doTaken together, we believe that the extravasation of monocytes from peripheral blood ascends, which promotes the extravasation of monocytes to the inflamed tissue so as to undergo further differentiation and function the clearance of pathogens.E. hellem stimulates the response of inflammatory monocytes, promotes proliferation of this set of cells in bone marrow, and ascends the extravasation of monocytes in the peripheral blood. Our findings also demonstrate the E. hellem infection would promote these monocytes differentiated into residential macrophage in the inflamed tissue.In this study, we found that the infection of + monocytes group are recruited to the peripheral tissue through blood circulation, and then further differentiated into activated macrophages. The derivative macrophages then express cell surface marker F4/80 [+ monocytes would gradually downregulate the expression of Ly-6C [+ monocytes act as inflammatory monocytes with the high capacity to migrate to the site of inflammation [Firmicutes, one of the major producers of butyrate, cooperates with the regeneration of bone marrow and promotes the differentiation of hematopoietic stem cells by providing the irons and metabolites [E. hellem infection can change the abundance of Firmicutes in microbiota [During infection, the Ly-6Cer F4/80 . In steaof Ly-6C , while uammation . Our finabolites . E. hellcrobiota . AccordiE. hellem infection. It could be the result of the decreasing of absolute number of monocytes due to increased extravasation. This inference may be supported by the study that concluded Leishmania major infection leads to the activation of bone marrow HSPCs and enhances myelopoiesis in the bone marrow, with a decreased absolute number of monocytes in the peripheral blood [Listeria monocytogenes infection, monocytic left shift occurs in the bone marrow at about 72 h [E. hellem, which needs further exploration.In the peripheral blood, we found the expression level of Ly-6C decreased after al blood ,28. In tout 72 h . Such a Encephalitozoon cuniculi up-regulated expression of CCL1, CCL2, CCL3, CCL7 and other chemokines [E. hellem infection and are consistent with the expression change patterns of Ly-6C on monocytes after infection. All indicate that, under the condition of E. hellem infection, bone marrow monocytes are prompted to proliferate and egress to blood driven by CCLs-CCR interaction, and this chemotaxis force drives more monocytes extravasation into inflamed tissues.The main driving force of monocyte egression and extravasation is the chemokine CCLs, such as CCL7 interacting with the CCR2 expressed on the cell surface . It is remokines . In thisE. hellem infection. In this study, we confirmed the upregulated expressions of F4/80 in small intestine of E. hellem-infected mice. The up-regulation of differentiated macrophage and the CCR2 together indicate that microsporidia infection stimulates more macrophages to this immune response, yet maybe later or during long-term latent infection, these immune cells would be hijacked to act as a “Trojan horse”, which had been found under the infection of Encephalitozoon cuniculi [The gastrointestinal tract is the major colonization site for microsporidia infection . Therefocuniculi ,31.More interestingly, the maturation and migration of dendritic cells are reported to be inhibited by microsporidia infection . TherefoE. hellem, host monocytes in the bone marrow proliferated in response to the infection. The extravasation of monocytes also increased significantly upon reaching the site of infection through peripheral blood. In this way, the differentiation of monocytes to phagocytic cells such as macrophages were promoted. Our study is the first investigation in a systemic view about monocytes proliferation and extravasation in responses to E. hellem infection.In this study, we found that during the infection course of


Taken together, HCC-targeted exosomes (ExoSP94-Lamp2b-RRM) could specifically deliver multi-siRNA to HCC tissues, enhance sorafenib-induced ferroptosis by silencing GPX4 and DHODH expression and consequently increase HCC sensitivity to sorafenib, which opens a new avenue for clinically overcoming sorafenib resistance from the perspective of ferroptosis.Sorafenib is one of the few effective first-line drugs approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common among individuals with HCC. Recent evidence indicated that the anticancer activity of sorafenib mainly relies on the induction of ferroptosis. Furthermore, in our study, genes that suppress ferroptosis, especially GPX4 and DHODH, were enriched in sorafenib-resistant cells and primary tissues and were associated with poor prognosis of HCC patients who received sorafenib treatment. Therefore, a new ferroptosis inducer comprising a multiplex small interfering RNA (multi-siRNA) capable of simultaneously silencing GPX4 and DHODH was created. Then, exosomes with high multi-siRNA loading and HCC-specific targeting were established by fusing the SP94 peptide and the N-terminal RNA recognition motif (RRM) of U1-A with the exosomal membrane protein Lamp2b. The results from the  Hepatocellular carcinoma (HCC) was ranked as the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related death worldwide in 2018 . Howeverc-) activity, downregulating GPX4 or DHODH expression, and accumulating reactive oxygen species (ROS)  . Recent ROS) (c- . MoreoveROS) c- , 8–11. T–11. Tc- SP94-lamp2b-RRM) with high tumor targeting ability and high multi-siRNA loading efficacy were constructed to deliver the multi-siRNA cargo. Using in vitro and in vivo models, we demonstrated that this tumor-targeting nanodrug (ExoSP94-lamp2b-RRM-multi-siRNA) could enhance sorafenib-induced ferroptosis and overcome sorafenib resistance, suggesting that it is a promising therapeutic strategy for treating sorafenib-resistant HCC.Herein, we first found that ferroptosis suppressor genes, especially GPX4 and DHODH, are enriched in sorafenib-resistant cells and primary tissues from patients and are associated with poor prognosis of HCC patients who receive sorafenib treatment. Then, we created a novel ferroptosis inducer comprising a multiplex small interfering RNA (multi-siRNA) suitable for simultaneously silencing GPX4 and DHODH. Then, exosomes (ExoThe antibodies used targeted the following proteins (dilutions used are included):GAPDH  from CWBIOTECH; F-actin  from YEASEN; U1A  from Proteintech; CD63  from Proteintech; CD9  from Proteintech; TSG101  from Proteintech; GPX4  from Proteintech; and DHODH  from Proteintech.The inhibitors used are as follows:in vitro assay and 30 mg/kg for the in vivo assay) and Ferrostatin-1 (HY-100579: 60 nM for the in vitro assay), and ferrostatin-1  were obtained from MedChem Express.Sorafenib . These cell lines were authenticated by the analysis of short tandem repeat (STR) profiles, and all of them matched those of the standard cell lines in the DSMZ data bank. These cells tested negative for cross-contamination of other human cells and mycoplasma contamination. The sorafenib-resistant HepG-2 cell line (HepG-2R) was generated by continuous treatment of HepG2 cells with sorafenib up to 10 μM and maintained as previously described . HepG-2,Briefly, the sequences for SP94 peptide (SFSIIHTPILPL), the CDS sequences of Lamp2b and the CDS sequences of the N-terminal RNA recognition motif (RRM) of U1-A were orderly arranged from N-terminal to C-terminal. Then the sequence encoding the above fusion protein was digested with BamHI-XhoI and cloned into pcDNA3.1(+) vector. The clones were confirmed by DNA sequencing and stored for the following experiments.For exosome isolation from HEK-293T cells, cells were transfected with control or SP94-Lamp2b-RRM expressing plasmids with Lipofectamine3000. 12 h later, cells were further cultured in DMEM with 10% exosome-depleted FBS  for 24–36 h. Then, the culture medium was precleared by centrifugation at 500×g for 15 min and then at 10000×g for 20 min. Exosomes were isolated by ultracentrifugation at 110000×g for 70 min at 4°C and washed in PBS using the same centrifugation conditions. The concentration of exosomes was determined by measuring total exosomal protein using a Pierce BCA Protein Assay Kit  according to the manufacturer’s instructions. NanoSight and transmission electron microscopy were used to determine the size distribution, concentration and morphology of the exosomes. For siRNA loading experiments, 100 nM corresponding siRNAs were transfected with Lipofectamine 3000. Twenty-four hours later, the cells were further cultured in DMEM with 10% exosome-depleted FBS for another 24 h, followed by exosome isolation.Cells were collected and fixed with 2.5% glutaraldehyde. Subsequently, cells were postfixed in 2% tetroxide and dehydrated through a series of gradient ethanol solutions. Samples were embedded in epoxy resin, cut into thin slices, and placed onto a nickel grid. Images were acquired using a Tecnai G2 Spirit transmission electron microscope (Thermo Fisher).A lipid ROS assay was performed as described previously. Briefly, cells were incubated with PBS containing 10 µM DCFDA dye in a cell culture incubator for 30 min. Cells were then collected and washed twice with PBS followed by resuspension in 200 µl of PBS. ROS levels were analyzed using a Beckman CytoFLEX system through the FITC channel.Analysis of lipid peroxidation was assessed by quantifying the MDA concentration in cell lysates using a Lipid Peroxidation MDA Assay Kit  in accordance with the manufacturer’s instructions.Six-week-old, male, athymic BALB/c nude mice were used. All animal experiments were carried out under protocols approved by the Animal Care and Use Committee of Fourth Military Medical University.in vivo tracking of exosomes, purified exosomes with the indicated modifications were labeled with the fluorescent dye DiR at a final concentration of 8 μM (Invitrogen). Labeled exosomes were collected by ultracentrifugation after washing with saline and stored in saline before use. Mice were injected with labeled exosomes (100 μg at the protein level in 100 μL) via tail vein injection. Mice were subjected to fluorescent living imaging 6 h after injection with an in vivo imaging system (IVIS lumina II).For 6 sorafenib-resistant HepG-2 cells stably expressing luciferase were surgically implanted into the left liver lobes of mice. Tumor growth was monitored by bioluminescence with an in vivo imaging system (IVIS lumina II). Two weeks after inoculation, mice were randomized to each group and began to receive different treatments. In the sorafenib treatment group, sorafenib (30 mg/kg) was given every 3 days. In the sorafenib and exosome combination treatment group, sorafenib was administered at the same dose, and 100 μg of the indicated exosomes (at the protein level) was injected via the tail vein 24 h after every sorafenib administration. At the end of the experiment, mice were sacrificed. Tumor bearing liver tissues and the main organs were collected for the following experiments.For orthotopic implantation, six-week-old male nude mice were anesthetized with 3% (w/v) pentobarbital sodium by intraperitoneal injection. Then, 2×102O2 in methanol for 10 min, the sections were washed three times in PBS and blocked with goat serum for 20 min. Then, they were incubated with primary antibodies in a humid container at 4°C overnight. After the addition of the PowerVision™ complex, tissue sections were incubated at 37°C for 20 min followed by treatment with DAB. PBS in place of primary antibody was used as a negative control.This experiment was performed as previously described . BrieflyP<0.05 was considered statistically significant. The statistical tests were two-sided.The data are presented as the mean ± s.e.m. from at least three independent experiments. Statistical analysis was performed using GraphPad Prism software. A random number table was used to randomize the mice into control and treatment groups. The numbers of mice were determined on the basis of our pretests and previous experience with similar experiments. A value of GPX4) resides in the center of a network that functions to prevent the accumulation of lipid hydroperoxides, thereby strongly suppressing ferroptosis  ; thus, wroptosis . InteresGPX4 and DHODH can directly remove the dangerous products of iron-dependent lipid peroxidation and consequently protect the cell membrane from damage; when GPX4 and DHODH expression and/or function are dysregulated, ferroptosis ensues  in the SP94-Lamp2b-RRM-functionalized exosomes treated group. By contrast, relatively low level of green fluorescence was observed in control exosomes treated cells up to 60 min, confirming the targeting ability of SP94-Lamp2b-RRM-functionalized exosomes to HCC cells (SP94-Lamp2b-RRM-multi-siRNA#1). The results showed that ExoSP94-Lamp2b-RRM-multi-siRNA#1 could significantly enhance the effect of sorafenib  can selectively deliver therapeutic cargos to human HCC cells with no obvious adverse effects. This is the first report of tumor-targeted exosome delivery of ferroptosis inducers, which might open a new avenue for the systematic use of ferroptosis inducers to treat cancer.Currently, the poor solubility and potential off-target toxicity to normal cells and tissues preclude the systematic use of traditional ferroptosis inducers in vivo –34. Nanoications –39. For  in vivo . Here, a in vivo . Our datSP94-Lamp2b-RRM) to deliver multi-siRNA, thus overcoming acquired resistance to sorafenib from the perspective of ferroptosis.In summary, this study firstly identified that ferroptosis suppressor genes GPX4 and DHODH were enriched in sorafenib-resistant HCC and associated with compromised therapeutic efficiency of sorafenib. In addition, we described the use of multi-siRNA to enhance sorafenib-induced ferroptosis by simultaneously knocking down GPX4 and DHODH. More importantly, we designed HCC-targeted engineered exosomes (ExoThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by Animal Care and Use Committee of Fourth Military Medical University.XL, QY, and RZ contributed equally to this work. YG, CZ, and YZ contributed to project conceptualization and supervision. YG, XL, QY, CL, XG, RZ, and KZ contributed to the investigation. WQZ, SW, QH, WL, and ML contributed to the data curation; YG and XL contributed to writing the original draft; YG, CZ, YZ, WZ, and XL contributed to writing, reviewing, and editing the manuscript; and YG, CZ, and SW contributed to funding acquisition. All authors contributed to the article and approved the submitted version.This work was financially supported by grants from the Hong Kong Scholar Program; the National Natural Science Foundation of China (NSFC), NO. 81902678, 81802632, 82072910; the Key Research and Development Program of Shaanxi Province (NO. 2017ZDCXL-SF-01-03); and the Young Elite Scientist Sponsorship Program by CAST (No. YESS20210177).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Radix Rehmanniae Praeparata  is widely used as primal medicine in Chinese herbal formula for the treatment of Alzheimer’s disease (AD). However, the underlying mechanism of RRP for AD remains unclear. The aim of this study was to investigate the therapeutic effect of RRP on intracerebroventricular injection of streptozotocin (ICV-STZ)-induced AD model mice and its potential mechanism. ICV-STZ mice were continuously gavaged with RRP for 21 days. The pharmacological effects of RRP were evaluated by behavioral tests, brain tissue H&E staining and hippocampal tau protein phosphorylation levels. The expression levels of insulin receptor (INSR), IRS-1, pSer473-AKT/AKT and pSer9-GSK-3β/GSK-3β proteins in hippocampal and cortical tissues were detected by Western-blot method. The 16S rRNA gene sequencing was used to analyze the changes of intestinal microbiota in mice. The compounds in RRP were analyzed by mass spectrometry and their binding ability to INSR proteins was detected by molecular docking. The results showed that RRP ameliorated cognitive dysfunction and neuronal pathological changes of brain tissue in ICV-STZ mice, reduced tau protein hyperphosphorylation, INSR, IRS-1, pSer473-AKT/AKT, and pSer9-GSK-3β/GSK-3β levels in hippocampal and cortical tissues. Meanwhile, RRP reversed ICV-STZ-induced dysregulation of intestinal microbiota in AD mice. Mass spectrometry analysis showed that the RRP consisted mainly of seven compounds, namely Acteoside (Verbascoside), 5-Hydroxymethyl-2-furaldehyde (5-HMF), Apigenin7-O-glucuronide, Icariin, Gallic acid, Quercetin-3β-D-glucoside, and Geniposide. Molecular docking results further indicated that the compounds in RRP have binding ability to INSR protein and potential multiple synergistic effects. RRP ameliorates cognitive dysfunction and brain histopathological changes in AD mice. The mechanism of RRP ameliorating AD may be related to the regulation of INSR/IRS-1/AKT/GSK-3β signaling pathway and intestinal microbiota. This study supports the potential anti-AD efficacy of RRP and initially reveals the pharmacological mechanism of RRP, providing a theoretical basis for further clinical application of RRP. Alzheimer’s disease (AD), the most common neurodegenerative disease, is one of the greatest medical challenges of this century and is becoming increasingly prevalent worldwide . The numClostridium butyricum  and can improve peripheral insulin resistance (The Chinese medicine Rehmannia (Sheng Dihuang in Chinese) is the dried root of the perennial herb Rehmannia glutinosa Libosch of the scrophulaceae family. Radix Rehmanniae Praeparata  is obtained by steaming Rehmannia with wine to modify its medicinal properties . RRP is od sugar  and enhaod sugar . Accordisistance . Also, Rsistance . Catalposistance . Our presistance . Howeversistance . The aimIn this study, we explored the efficacy of RRP using ICV-STZ-induced mice and examined the expression levels of INSR/IRS-1/AKT/GSK-3β signaling pathway related proteins in brain tissue. Meanwhile, the intestinal microbiota of mice in each group was analyzed. In addition, we analyzed the compounds in RRP and assessed the interaction of compounds with INSR proteins by molecular docking. The results showed that RRP has anti-AD effect and the mechanism maybe related to the regulation of INSR/IRS-1/AKT/GSK-3β signaling pathway and intestinal microbiota.Ser404-tau, IRS-1, AKT, pSer473-AKT, GSK-3β, and pSer9-GSK-3β , Horseradish peroxidase (HRP)-linked secondary antibodies against rabbit and mouse IgG of the primary antibodies  and Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 .RRP extracted powder  was purchased from Shaanxi Sypal Bio-technique Co., LTD. The main materials involved in this study included STZ , isoflurane , Hematoxylin-Eosin Staining Kit , DAPI , primary antibodies β-Actin , INSR , p−1 STZ, which is an optimized animal model in AD studies resulting in hyperphosphorylation of tau (-1) was dissolved in sodium citrate  prepared before injection. For sodium citrate buffer preparation, 2.1 g citric acid (FW: 210.14) was added to 100 mL of distilled water to form liquid A, and 2.94 g sodium citrate (FW: 294.10) was added to 100 mL of distilled water to form liquid B. When used, liquid A and liquid B were mixed 1:1 to form sodium citrate buffer. The mice were fixed in a brain stereotaxy instrument. After anaesthetized by anesthesia machine, the anterior fonthalle was exposed through a median incision at the top of the lateral ventricle. The right lateral ventricle was slowly injected with STZ for 5 min and left it for another 5 min before slowly withdrawing the needle. All procedures were performed in a sterile environment. The wounds were sutured after antiseptic treatment with penicillin. The sham-control group (control group) was injected with an equal volume of sodium citrate buffer. Control group, model group and RRP-treated group were set up with 10 mice in each group. RRP extracted powder was dissolved in saline and mice were administered intragastrically with a dose of 400 mg kg−1 d−1 for 21 days according to the suggestion of Pharmacopoeia of the People’s Republic of China and the dose optimization was performed in the previous study  were purchased from Weitong Lihua Laboratory Animal Technology Co., LTD. . The experimental animal license number is SCXK (jing) 2021-0006. The animal room was maintained at room temperature of (22 ± 2)°C, humidity of 60%, 12:12 h light/dark cycle conditions, free diet and water. The animal experimental design was in accordance with the requirements of the Experimental Animal Ethics Committee of Henan University of Chinese Medicine (DWLLGZR202202147). After 1 week of adaptive feeding, AD models were prepared by lateral ventricular injection of 3 mg kgn of tau . STZ  staining was performed to compare the histopathological changes in each group. All brain tissues were paraffin-embedded and sectioned according to the conventional method . 5 μm thSer404-tau and Alexa Fluor 594 goat anti-mouse IgG (H + L) were used as primary and secondary antibodies, respectively, and finally nuclear staining was performed with DAPI  and species analysis  were used to evaluate the abundance and diversity of the microbiota. Beta diversity analysis based on weighted UniFrac was used to evaluate the differences in microbiotic structure between samples . Genus-level species composition heat maps were drawn for species clustering, in which samples were clustered according to the Euclidean distance of species composition data in UPGMA and ranked according to the clustering results. Statistical analysis of phylum and class level abundance among different groups was performed based on ASVs/OTU sequence abundance. 16S rRNA gene sequencing was performed by Suzhou PANOMIX Biomedical Tech Co., LTD.  platform. The amplicon sequence variants (ASVs)/operational taxonomic units (OUT) were clustered using the DADA2 method combined with Vsearch method quality control to obtain the amplicon sequence variants (ASVs)/OUT abundance table with 100% similarity. Alpha diversity analysis , and the evasion latency of the RRP-treated group was significantly shorter from day 4 compared with the model group (p < .05)   (p < .05), and significantly increased in the RRP-treated group (p < .01) (p < .05), while the number of grooming was significantly increased in RRP-treated group compared to the model group (p < .05) (p < .01), and significantly higher in the RRP-treated group compared to the model group (p < .05)  . The resp < .05) , and elap < .05) . Open fip < .01) . The nump < .05) . Figure p < .05) . Spontanp < .05) .As shown in Ser404-tau protein expression levels were significantly elevated in the model group compared with the control group (p < .01), and pSer404-tau protein levels were significantly lower in the RRP-treated group compared with the model group (p < .01) , while a small amount of pSer404-tau protein was present in the brain tissue of RRP-treated mice, which was significantly lower compared to the model group (p < .05) .Western-blot results showed that pp < .01) . Similarp < .01, p < .05) and cortical  of mice in the model group compared to the control group, while the expression levels of INSR, IRS-1 proteins were significantly increased in hippocampal  and cortical  of mice in the RRP-treated group compared to the model group. Similarly, expression ratios of pSer473-AKT/AKT, and pSer9-GSK-3β/GSK-3β were significantly decreased in hippocampal  and cortical  of mice in the model group compared to the control group, while the ratios were significantly increased in hippocampal  and cortical  of mice in the RRP-treated group.As shown in p > .05), while Simpson and Shannon values were significantly decreased . Compared with the model group, the Chao1, Simpson, and Shannon values were increased in the RRP group with no significant difference (p > .05). PCOA and NMDS analysis of β-diversity showed that the colony structure was independently separated between the control and model groups, while there was an overlap between the control and RRP groups  and Firmicutes and Bacill were significantly increased  in model group compared to control group. After RRP treatment, the levels of Bacteroidetes, Bacteroidia were significantly increased significantly , while the levels of Firmicutes and Bacill were significantly decreased significantly in mice of RRP group compared to model group .The structure of compounds was identified by mass spectrometry. Ten compounds in the sample had a combined score of >80 in the mzCloud best match, and seven of which were reported in the RRP. These seven compounds were Acteoside (Verbascoside), 5-Hydroxymethyl-2-furaldehyde (5-HMF), Apigenin7-O-glucuronide, Icariin, Gallic acid, Quercetin-3β-D-glucoside, and Geniposide respectively. The chemical formula and structure are shown in −1 eight hydrogen bonds were formed with GLU1094, ASN1097, ARG1089, ASP1083, and WER1086. Different compounds bind to INSR proteins at different sites. The number of hydrogen bonds formed by the compounds in RRP with INSR proteins, the minimum binding energy and the binding sites are shown in The molecular docking results showed that compounds in RRP could bind to INSR protein, among which the binding energy of Icariin to INSR protein was −9.7 kcal molThe pathogenesis of AD is complex and clinical medications are lacking . TraditiRRP is widely used as primal medicine in Chinese herbal formula for the treatment of AD or diabetes, for example, Qi Fu Yin, Qidan Dihuang decoction, Zhibai Dihuang Wan, Dihuang Yinzi, and Liuwei Dihuang . SeveralSer404-tau protein in the brain tissue of AD mice, indicating that RRP improved the pathology of AD.In the water maze experiment, the escape latency is the time required for mice to successfully find the platform for the first time after each entry into the water, and a short latency period predicts good learning memory ability of mice. The longer the time elapsed in the target quadrant and the greater the number of crossings, the better the spatial learning and memory ability of the animal. The open field experiment can be used to observe the autonomous locomotor ability of experimental animals. The total distance moved and the number of grooming are the stress behaviors produced in response to the new and different environment, indicating that the mice are more excited and have better autonomous locomotor ability at this time. The alternation triplet in the Y-maze experiment indicated the spatial exploration ability of mice, and the high alternation rate meant that the mice had better spatial exploration ability. The results of behavioral tests showed that RRP treatment decreased the escape latency and increased the elapsed time in targeted quadrant and the number of crossings in water maze experiment, increased the total distance and grooming number in open field test and alternation triplet in Y-maze experiment in AD mice, indicating that RRP improved the cognitive dysfunction of AD mice. H&E staining showed that pathological damage in the brain was reduced in the RRP-treated group of mice, suggesting a neuroprotective effect of RRP in AD mice. AD is the most common neurodegenerative disease and hyperphosphorylation of tau protein is considered as one of the pathological markers . Ser404 Ser473-AKT/AKT, and pSer9-GSK-3β/GSK-3β in brain tissue, indicating that RRP restored the insulin signaling pathway in the brain of AD mice. It is not clear which components of RRP can regulate the insulin signaling pathway. Seven compounds were identified from the extracted powder of RRP by mass spectrometry analysis. According to the reports, Acteoside (Insulin signaling is considered to be a common link connecting many other AD hypotheses, which is a hot spot of research for anti-AD drug development . In the cteoside , 5-HMF (cteoside , Icariincteoside , Gallic cteoside , and Gencteoside  are poteIntestinal microbiota imbalance is closely associated with AD pathogenesis . Recent In summary, it is concluded that RRP significantly improved cognitive dysfunction and brain pathological changes in AD mice. The mechanism of RRP improvement in AD may be related to the regulation of INSR/IRS-1/AKT/GSK-3β signaling pathway and intestinal microbiota. This study provides a theoretical basis for the clinical application of RRP. However, this study is limited in that only one animal model of AD was selected for study and no reverse validation was performed. In the future, transgenic mouse models of AD and reverse validation at the cellular level is need to be tested. In addition, the further identification of active ingredients in RRP will provide more scientific basis for the development of new anti-AD drugs.


Flaviviruses, represented by Zika and dengue virus (ZIKV and DENV), are widely present around the world and cause various diseases with serious consequences. However, no antiviral drugs have been clinically approved for use against them. Azelnidipine (ALP) is a dihydropyridine calcium channel blocker and has been approved for use as an antihypertensive drug. In the present study, ALP was found to show potent anti-flavivirus activities in vitro and in vivo. ALP effectively prevented the cytopathic effect induced by ZIKV and DENV and inhibited the production of viral RNA and viral protein in a dose-dependent manner. Moreover, treatment with 0.3 mg/kg of ALP protected 88.89% of mice from lethal challenge. Furthermore, using the time-of-drug-addition assay, the enzymatic inhibition assay, the molecular docking, and the surface plasmon resonance assay, we revealed that ALP acted at the replication stage of the viral infection cycle by targeting the viral RNA-dependent RNA polymerase. These findings highlight the potential for the use of ALP as an antiviral agent to combat flavivirus infections. Flaviviridae, a class of single-stranded, enveloped RNA viruses with hundreds of members, such as DENV, ZIKV, Japanese encephalitis virus (JEV), West Nile virus (WNV), and yellow fever virus (YFV). DENV is the most globally widespread arbovirus [Aedes mosquitoes, but other transmission routes, such as mother-to-child transmission and sexual contact, have also been reported [Flaviviruses, represented by dengue virus (DENV) and Zika virus (ZIKV), belong to the family rbovirus . There arbovirus . Althougrbovirus ,4. ZIKV reported . Recent reported ,7,8. FlaDihydropyridine calcium channel blockers (DHP-CCBs), a series of compounds approved for the treatment of cerebrovascular diseases, have exhibited favorable efficacy against various virus infections . AmlodipIn our study, ALP exhibited potent antiviral activity against ZIKV and DENV in vitro and in vivo. Mode-of-action studies have revealed that ALP directly targets the viral RNA-dependent RNA polymerase (RdRp) and inhibits viral replication. These findings should help in constructing a novel antiviral approach with DHP-CCB distinct from that described in previous reports, and highlights the potential for the use of ALP as an anti-flavivirus drug candidate.2. Flaviviruses including ZIKV (SMGC-1 strain) and DENV  strain) were passaged and propagated as previously described [50).Vero (Cat #CCL-81) cells were purchased from the American Type Culture Collection . BHK cells were purchased from the National Infrastructure of Cell Line Resource, China. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin at 37 °C and in 5% COescribed . The virAzelnidipine , 7-deaza-2′-C-acetylene-adenosine , 2′-C-methyladenosine , and 3′-dATP  were dissolved in DMSO and stored at −20 °C until used.3 or 1 × 104 cells per well, and incubated at 37 °C overnight. The cells were treated with threefold serially diluted ALP and 100 × TCID50 viruses until an obvious CPE was observed. The cell viability of each group was then tested in accordance with the procedure of the CellTiter-Glo Luminescent Cell Viability kit. The half-maximal inhibitory concentration (IC50) value of ALP was fitted and calculated using the Origin 9.0 software. The assessment method for the 50% cytotoxic concentration (CC50) was similar to that for IC50, except that the virus inoculum was replaced with DMEM containing 2% FBS.BHK and Vero cells were used for the evaluation of ALP’s antiviral effects against DENV and ZIKV, respectively. The cytopathic effect (CPE) protection assay was performed according to previously reported methods . Briefly® Mini Kit and quantified using qRT-PCR, as previously reported [For the virus yield reduction assay, the BHK or Vero cells were seeded in a 12-well plate and cultured overnight, and they were then inoculated with gradient-diluted ALP in the presence of the viruses (multiplicity of infection (MOI) = 0.05). After 72 h of incubation, the cellular RNA was extracted using the RNeasyreported . The vir5 cells/well in a 12-well plate and cultured at 37 °C overnight. The cells were inoculated with ZIKV or DENV for 0 to 2 h, and ALP (6 μM) or the control drug NITD008 (5 μM) was added at four intervals, which shows different stages of the virus infection cycle. The cells in each group were washed three times with DMEM containing 2% FBS after the treatment. The intracellular viral RNA was extracted at 24 h.p.i. and determined using the qRT-PCR method.A time-of-drug-addition assay was performed as previously described with some modifications ,18. BrieThe ZIKV replicon inhibition assay was carried out as described previously . ConflueZIKV forward primer—GGTCAGCGTCCTCTCTAATAAACG;Reverse primer—GCACCCTAGTGTCCACTTTTTCC;Probe—AGCCATGACCGACACCACACCGT;DENV forward primer—AGGTCGGATTAAGCCATAGTACG;Reverse primer—TGGCCTGACTTCTTTTAACGTC;Probe—AAAAACTATGCTACCTGTGAGCCCCGTCC.The total cellular RNA was harvested, and the viral RNA was quantified using qRT-PCR according to the procedure of the One Step PrimeScript RT-PCR kit. The specific primers and probes used for ZIKV and DENV assessment were as follows: The viral RNA copies were determined from the cycle threshold (Ct) value of each sample, with reference to the known copy number standard curve.50 values of the test compounds using the Origin 9.0 software.The DENV RdRp protein was expressed and purified, and a real-time fluorescence-based RdRp enzymatic system was constructed and optimized as described previously ,21. To eThe SPR assay was carried out using the Biacore 8K  instrument to explore the interaction of DENV-RdRp protein with ALP. RdRp proteins in 10 mM sodium acetate were immobilized on the CM5 chip. Then, gradient-diluted ALP (50–3.13 μM) in a running buffer consisting of 1 × PBS with 0.05% Tween-20 and 5% DMSO at pH 7.4 flowed over the sensor chip surface. The response units were recorded and fitted using the Biacore Evaluation Software.The crystal structures of DENV RdRp protein (PDB: 5K5M) and ZIKV RdRp protein (PDB: 6LD5) were obtained from the Protein Data Bank (PDB) database. The “Prepare Protein” module of the Discovery Studio 4.5 software was used to remove the conformation of the nontarget protein and supplement the incomplete amino acid residues. ALP was prepared using the “Prepare Ligands” module to obtain an effective three-dimensional conformation, and then docked with CDOCKER for target proteins. The key interactions and amino acid residues were analyzed using the PyMOL 2.5 software.4 PFU of virus particles, followed by the administration of ALP  or the vehicle at 4 h post-infection. After that, the mice in each group were i.p. treated once daily for 9 consecutive days. The survival and bodyweight of the infected mice were recorded for 21 days.The animals used in this study were specific pathogen-free grade, and all the experiments and operations were reviewed and approved by the Institutional Animal Care and Use Committee of Beijing Institute of Pharmacology and Toxicology. One-day-old ICR suckling mice were used to evaluate the in vivo anti-ZIKV efficacy of ALP. Briefly, each suckling mouse was intraperitoneally (i.p.) injected with ZIKV at a volume of 25 μL containing 1.3 × 10t-test or one-way analysis of variance was used for data analysis. The survival curve was determined using the log-rank test. The results are expressed as the means ± standard error of mean (SEMs). Considered statistically significant was p < 0.05.Statistical analyses were performed using the GraphPad Prism 7 software. An unpaired two-tailed Student’s 50 values were 1.68 ± 0.50 μM and 0.76 ± 0.17 μM for ZIKV and DENV, respectively. Then, we performed qRT-PCR and immunofluorescence assays to assess the effects of ALP on the yields of viral RNA and proteins. As shown in To explore the in vitro anti-flavivirus potential of ALP A, two re50 values of 1.67 ± 0.01 μM and 2.70 ± 0.06 μM, respectively. Collectively, these data suggest that ALP mainly acted at the post-entry stage of flavivirus infection.To determine the mechanism of action of ALP, a time-of-drug-addition assay was first conducted to determine the specific stage at which ALP acted, as shown in the schematic illustration in 50 values of ALP and the positive control compound 3′-dATP were determined to be 6.72 ± 4.69 μM and 30.09 ± 8.26 μM, respectively  is the key viral protein with enzymatic activity and is responsible for the replication of the viral genome. Considering that the RdRp protein is a key enzyme in the viral replication process, we hypothesized that ALP might exert its antiviral effects through the inhibition of viral RdRp protein. To this end, a real-time fluorescence-based enzymatic system using the DENV RdRp protein was first constructed and optimized according to previously reported methods . We thenectively B. To furin silico ligand-docking assay was performed using the solved crystal structure of the flavivirus RdRp protein. The RdRp protein consisted of three subdomains, namely, the palm, thumb, and fingers subdomains. To date, two modes of small molecular inhibitors’ binding with the flavivirus RdRp protein have been reported [To further explore the mode of ALP’s binding with the flavivirus RdRp protein, an reported ,23,24. Freported ,26. The 4 PFU per mouse of ZIKV. Mice in different groups were administered the vehicle or ALP at a dose of 0.03 or 0.3 mg/kg at 4 h post-infection, followed by the same dosage once daily for 9 consecutive days. As shown in To evaluate the in vivo antiviral effects of ALP, we first utilized the ZIKV lethal infection model to evaluate the anti-ZIKV potential of the tested compound. One-day-old ICR suckling mice were inoculated with 1.2 × 10To further determine the protective effect of ALP, we euthanized and dissected the mice on the third day after infection and examined the viral load in different tissues. The results show that, after challenge, the ALP-treated group showed significantly reduced viral loads in the brain and liver compared with the untreated control mice. However, no significant difference of viral loads in the spleen between the control and the treatment was observed C. Due to50 values of 1.68 ± 0.50 μM and 0.76 ± 0.17 μM, respectively. Additionally, the yields of the viral RNA and proteins in the ALP-treated groups were decreased in a dose-dependent manner. To explore the mechanism of ALP’s action against ZIKV and DENV, we first carried out a time-of-addition assay, and the results revealed that ALP acted at the post-entry stage of flavivirus infection, which is consistent with the results obtained via the replicon inhibition assay. Considering that the RdRp protein plays a key role in intracellular viral replication, we constructed a real-time fluorescence-based enzymatic system and evaluated the effects of ALP against the viral RdRp protein. To our surprise, a potent inhibitory effect (IC50 = 6.72 ± 4.69 μM) was observed in the RdRp catalytic assay in accordance with the results obtained in the in vitro antiviral assays and the SPR assay. Furthermore, the results of the ligand-docking assay indicated that ALP may bind at an allosteric site anchored by the priming loop, specifically the N pocket of the flavivirus RdRp protein. Flaviviruses transmitted through multiple routes could lead to the development of a variety of diseases with serious consequences . However2+ concentrations [2+ influx using different WNV strains and different cell types. Treatment with BAPTA or EGTA, an extracellular calcium chelator, reduced the Ca2+ influx and decreased the viral yield early in infection. They suggest that WNV infection leads to the early persistent activation of FAK, ERK1/2, and PI3K/Akt in mammalian cells [2+ signaling [It has been demonstrated that host cell dysfunction following viral infection is accompanied by abnormal intracellular Catrations . One resan cells . Howeverignaling . Wang etignaling . Interesignaling . In thisAt present, there have been no reports on the use of azelnidipine for the treatment of infectious diseases. However, benidipine and nifedipine, two calcium channel blockers, inhibited SFTSV replication in vitro . A retro


Subjective and objective cognitive dysfunction are reported after COVID-19 but with limited data on their congruence and associations with the severity of the acute disease. The aim of this cohort study is to describe the prevalence of subjective and objective cognitive dysfunction at three and six months after COVID-19 and the associations of subjective cognitive symptoms and psychological and disease-related factors.We assessed a cohort of 184 patients at three and six months after COVID-19: 82 patients admitted to the Intensive Care Unit (ICU), 53 admitted to regular hospital wards, and 49 isolated at home. A non-COVID control group of 53 individuals was included. Demographic and clinical data were collected. Subjective cognitive symptoms, objective cognitive impairment, and depressive and post-traumatic stress disorder (PTSD) symptoms were assessed.At six months, subjective cognitive impairment was reported by 32.3% of ICU-treated, 37.3% of ward-treated, and 33.3% of home-isolated patients and objective cognitive impairment was observed in 36.1% of ICU-treated, 34.7% of ward-treated, and 8.9% of home-isolated patients. Subjective cognitive symptoms were associated with depressive and PTSD symptoms and female sex, but not with objective cognitive assessment or hospital metrics.One-third of COVID-19 patients, regardless of the acute disease severity, reported high levels of subjective cognitive dysfunction which was not associated with results from objective cognitive screening but with psychological and demographic factors. Our study stresses the importance of thorough assessment of patients reporting long-term subjective symptoms, screening for underlying mental health related factors such as PTSD or depression. COVID-19 patients have commonly reported subjective cognitive complaints  with as Our aim was to assess subjective and objective cognitive dysfunction up to six months after COVID-19 in three different levels of acute disease severity: patients treated in the ICU or regular hospital wards or isolated at home. We examined the prevalence of subjective and objective cognitive impairment and the associations of subjective cognitive impairment to objective cognition, symptoms of depression and PTSD, and demographic and disease-related factors at three and six months after COVID-19.22.1ClinicalTrials.gov (NCT04864938). The Ethics Committee of Helsinki University Hospital (HUS-1949-2020) approved the protocol. All participants gave written informed consent to the study and the principles of the Declaration of Helsinki were followed.This is a post hoc study of the RECOVID project, a multidisciplinary, single-center, prospective cohort study. The study protocol has been previously described . The stuWe assessed COVID-19 patients in three groups of acute disease severity . A contr2.2The main outcomes were the prevalence of subjective and objective cognitive impairment at 3 and 6 months after COVID-19 in three groups of acute disease severity compared to non-COVID controls. Secondary outcomes were the associations of subjective cognitive symptoms with objective cognitive impairment, demographic and disease-related factors, and depressive and PTSD symptoms.We evaluated subjective cognitive impairment with the A-B Neuropsychological Assessment Schedule (ABNAS) questionnaire , a scaleWe screened objective cognitive functioning with Montreal Cognitive Assessment (MoCA). The telephone-based version (t-MoCA)  was usedWe assessed post-traumatic stress disorder (PTSD) symptoms with the Impact of Event Scale-6 (IES-6), an abbreviated 6-item version of the IES-Revised (IES-R) that uses a five-point Likert scale ranging from 0 to 4 . The scoWe evaluated depressive symptoms with the two-question screening method (Depression screen)  at threeWe retrieved demographic information and clinical variables  from medical records. Education level was reported by the patient.2.3n (percentage). Group differences were assessed with ANOVA for normally distributed continuous variables, Kruskal-Wallis for non-normally distributed variables, and χ2 test for categorical variables. Changes in the frequency of impairment in follow-up were assessed with McNemar tests. Because of the differences in age and education levels in the groups, we used additional age- and education-adjusted ANCOVAs with Tukey's post hoc tests to assess differences in MoCA scores. We transformed all ABNAS subscale scores to a range of 0–3 to enable comparison of subscales with differing numbers of items and assessed group differences with ANOVA.We provide descriptive statistics for the demographic and clinical characteristics of each group and report the total sample size available for each data element. Sample size was based on power calculations for finding group differences which indicated that n > 50 would be required for each group. Continuous variables are presented as mean (SD) or median (interquartile range [IQR]) and categorical variables as We assessed the correlations of subjective cognitive symptoms with depressive and PTSD symptoms, demographic factors, hospital metrics, and objective cognitive impairment with Pearson's or Spearman's r correlations. We then used partial Pearson correlation coefficients controlling for age, education, and IES-6 to assess adjusted associations between subjective cognitive symptoms and objective cognitive performance. Finally, we used Venn diagrams to illustrate the overlap of objective cognitive dysfunction, subjective cognitive symptoms, and PTSD symptoms.p < .05 and all tests were two-tailed. The Bonferroni correction and the Benjamini-Hochberg adjusted false discovery rate (FDR) were used to control for multiple comparisons. No imputation was performed for missing data. Data were analyzed using IBM SPSS Statistics for Mac Version 27  and Jamovi version 2.2 .Statistical significance was set at 33.1p < .001). The ICU group had less education than the WARD (p = .002), HOME (p < .001), and CONTROL (p < .001) groups. The CONTROL group had 50.9% men and a mean age of 54.9 (12.3) years. Demographic characteristics, comorbidities, and hospital metrics are presented in The mean age of the COVID-19 patients was 53.4 (SD 12.8) years and 44% were men. The HOME group was younger than the ICU, WARD, and CONTROL groups  and language (p < .001), all patient groups reported more symptoms than controls  reported high levels of subjective cognitive symptoms (ABNAS score >15) at three months and 34.2% (53/155) at six months compared to 8.0% (4/50) of the CONTROL group. The frequencies of high ABNAS scores remained the same at three and six months . There wcontrols . In slow3.3p = .011, η2 0.06). The HOME group scores were higher than ICU (p = .042) and WARD (p = .009) groups. Age  and education  were significantly related to t-MoCA score. After adjusting for age and education, a significant group effect remained  but no significant post hoc pairwise differences were found after adjusting for multiple comparisons.The 3-month cognitive assessment took place on average 108 (SD 18) days after hospital discharge for ICU and 152 (41) days for WARD patients and 181 (29) days after positive COVID-19 test result for HOME patients. Cognitive impairment was observed in 77 of 151 (51.0%) patients . A statip < .001, η2 0.096). In the HOME group, scores were higher than in ICU (p < .001), WARD (p < .001), and CONTROL (p = .003) groups. Age  and education  were significantly related to MoCA score. After adjusting for age and education, differences in MoCA scores were statistically significant  with the HOME group having higher scores than the WARD (p = .006) and CONTROL (p = .024) groups.The 6-month cognitive assessment occurred on average 182 (18) days after hospital discharge for ICU and 198 (17) days for WARD patients and 220 (34) days after positive COVID-19 test result for HOME patients. Cognitive impairment was observed in 47 of 166 (28.3%) patients and 15 of 48 (31.3%) controls . The fre3.4IES-6 scores of >1.75 were found in 27.7% of patients at 3 months and 19.6% at 6 months . IES-6 sp = .034). A statistically significant difference was detected in the ABNAS score in the ICU group  but not in the WARD or HOME groups. At six months, there were no sex differences in ABNAS scores in patient groups or controls.Age and education did not correlate with ABNAS scores . Sex wasp = .431) and IMV  were not associated with ABNAS.No correlations were found between the length of hospital stay and ABNAS. In ICU patients, delirium . No statistically significant partial correlations between MoCA and ABNAS were found at 3 or 6 months in the ICU, WARD, or HOME groups, or in the group of all COVID-19 patients combined.There was a correlation with CU group . BecauseThe proportions of patients with significant subjective and objective cognitive symptoms and PTSD symptoms are described in Venn diagrams .Fig. 3Ve4In this prospective single-center study of 184 COVID-19 patients and 53 controls we observed a poor congruence of subjective and objective cognitive function test results and an association of subjective cognitive impairment with depressive and PTSD symptoms in a six-month follow-up. Over one-third of patients with COVID-19, regardless of the acute disease severity, reported high levels of subjective cognitive impairment in a six-month follow-up. Objective cognitive impairment was observed in more than half of ICU and WARD patients and one-third of HOME patients at 3 months. At 6 months the cognitive performance of all three COVID-19 groups was similar (ICU and WARD) or better (HOME) than non-COVID controls. The frequency of objective but not subjective cognitive impairment decreased from 3 to 6 months. Depressive and PTSD symptoms were equally reported by all COVID-19 groups. Subjective cognitive symptoms were associated with depressive and PTSD symptoms, and in the ICU group, female sex. No associations were found between subjective cognitive impairment and objective cognitive impairment, length of hospital stay, delirium, IMV, age, or education.The proportion of high levels of subjective cognitive symptoms in our study was approximately 30–40%, similar across the severity of acute disease, and stable in follow-up. Our findings differ from some studies where patients with more severe COVID-19 have reported more subjective symptoms  but are COVID-19 patients reported more subjective cognitive symptoms than controls especially in the domains of fatigue, slowing, and language. Fatigue has been one of the most reported symptoms after COVID-19 . An inteIn objective cognitive screening at three months, more than half of ICU and WARD and almost one-third of HOME patients had t-MoCA scores in the impaired range. The percentages of impaired MoCA scores decreased in follow-up. At six months, the rates of impaired cognitive performance in the ICU and WARD groups correspond to the findings of previous studies of hospitalized COVID-19 patients . HoweverSubjective cognitive symptoms were not associated with objective cognitive screening, length of hospital stay, delirium, or IMV. Some previous studies have found subjective cognitive complaints to correlate with objectively measured cognitive impairment , 2022 whPsychological factors may also have a role in persisting subjective cognitive complaints after COVID-19 . In our The strengths of our study include using a non-COVID control group which allows comparison to individuals who have been exposed to the indirect, societal factors associated with the pandemic. This is one of the first studies, to our knowledge, to investigate objective and subjective cognitive outcomes in several levels of COVID-19 severity. Additionally, the multidisciplinary approach allowed a comprehensive examination of clinical, cognitive, and psychological factors.There are several limitations to this study. First, the home-isolated group was the youngest and predominantly female, whereas the ICU group was predominantly male with the lowest education level, reflecting the risk factors for severe COVID-19 infection. There is a rather large sex difference in the home-isolated group with a high number of female subjects. In previous research, females have reported more long-term COVID-19 symptoms than males, which may have several possible explanations such as sex-related immune system differences or gender-related social factors . We cann5In conclusion, long-term subjective cognitive impairment and psychological symptoms were present in COVID-19 patients regardless of the severity of the acute disease. Subjective cognitive symptoms were associated with depressive and PTSD symptoms and female sex, but not with objective cognitive screening. Objective neuropsychological assessment and self-reported measures of cognition seem to assess different constructs, although providing valuable information on different aspects of functioning. It is important to thoroughly assess patients reporting long-term subjective symptoms and screen for underlying mental health related factors such as depression or PTSD.Helsinki University Hospital government funding for university level research (TYH2021310) and Nordforsk. R. Pihlaja was supported by the 10.13039/501100003125Finnish Cultural Foundation (grant number 00220808), Neurocenter, Helsinki University Hospital and the Department of Anaesthesiology, Intensive Care and Pain Medicine, Helsinki University Hospital. No other disclosures are reported. The sources of funding had no role in study design, the collection, analysis, and interpretation of data, the writing of the report, and the decision to submit the article for publication. Open access funded by Helsinki University Library.This work was supported by 10.13039/501100003125Finnish Cultural Foundation. Riikka Pihlaja reports financial support was provided by 10.13039/100008376HUS Helsinki University Hospital. All authors report financial support was provided by Nordforsk. All authors report financial support was provided by Helsinki University Hospital. Johanna Hastbacka reports a relationship with Paion AG that includes: consulting or advisory.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Riikka Pihlaja reports financial support was provided by


Xanthomonas oryzae pv. oryzicola (Xoo) is a plant pathogenic bacterium that can cause rice bacterial blight disease, which results in a severe reduction in rice production. Antimicrobial-dependent microbial controlling is a useful way to control the spread and outbreak of plant pathogenic bacteria. However, the abuse and long-term use of antimicrobials also cause microbial antimicrobial resistance. As far as known, the mechanism of antimicrobial resistance in agricultural plant pathogenic bacteria still lacks prospecting. In this study, we explore the mechanism of Zhongshengmycin (ZSM)-resistance in Xoo by GC-MS-based metabolomic analysis. The results showed that the down-regulation of the TCA cycle was characteristic of antimicrobial resistance in Xoo, which was further demonstrated by the reduction of activity and gene expression levels of key enzymes in the TCA cycle. Furthermore, alanine was proven to reverse the ZSM resistance in Xoo by accelerating the TCA cycle in vivo. Our results are essential for understanding the mechanisms of ZSM resistance in Xoo and may provide new strategies for controlling this agricultural plant pathogen at the metabolic level. Xanthomonas oryzae pv. oryzicola (Xoo) is a rod-shaped Gram-negative bacterium that can infect rice leaves through wounds or stomata, thereby causing bacterial blight (BB), one of the most devastating diseases of rice [Natural catastrophes, pests, and plant pathogens threaten the production of food crops worldwide, with diseases caused by plant pathogens accounting for 16% of global crop yield losses . Xanthom of rice . Low con of rice . Present of rice ,5,6,7.Nilaparvata lugens [Zhongshengmycin (ZSM herein) is a type of N-glucosides agricultural antimicrobial developed by the Chinese Academy of Agricultural Sciences Institute of Biological Control, which works by interrupting the synthesis of protein peptide bonds of pathogenic microorganisms to inhibit the growth of bacteria and fungal mycelium and prevents the germination of spores . ZSM hasa lugens ,9,10. Hoa lugens . For exaa lugens ,13. In ra lugens . TherefoEscherichia coli [Vibrio alginolyticus to ceftazidime [E. coli at sub-MIC doxycycline [The metabolome examines the overall metabolic level of a biological system under specific conditions, and it is the chemical entity transformed in the metabolic process of a physical system . It can hia coli ; triclostazidime ; and allycycline . Hence, ZS, respectively). We found that down-regulation of the TCA cycle may be the critical metabolic characteristic involved in the resistance to ZSM in Z173-RZS. In addition, exogenous alanine enhances the controlling effect of Xoo against ZSM by promoting the TCA cycle. Our results are important for understanding the mechanism of resistance to ZSM in Xoo and provide a novel approach to restoring the sensitivity of drug-resistant Xoo to ZSM.This study used GC-MS-based metabolomics to characterize the metabolomic changes in ZSM-sensitive/resistant Xoo strains  were detected through the Mann-Whitney U Test by SPSS analysis software, with the asymptotically significant eat maps A. The Z-maps ), we further analyzed the metabolic differences between Z173-S and Z173-Rzs. In total, 80 DMs were enriched in 41 metabolic pathways, and the 8 key metabolic pathways were identified as significantly involved pathways with a p < 0.05 , α-ketoglutarate dehydrogenase (α-KGDH), and pyruvate dehydrogenase (PDH) were all significantly decreased in Z173-Rh Z173-S A. In add73-S  is a pathogen that causes Xanthomonascampestrispv. oryzae. With the preventive use of antimicrobials, the emergence of drug-resistant bacteria of Xoo has occurred. Restoring the sensitivity of drug-resistant bacteria to antimicrobial agents is crucial for the sustainable development of agriculture. Therefore, there is an urgent need to better understand the mechanism of antimicrobial resistance in resistant bacteria and develop new drug treatment strategies. In recent years, studying drug resistance from the perspective of metabolism has attracted attention and research. Aros-Calt used LC-MS to study the methicillin-resistant mechanism of Staphylococcus aureus [Salmonella typhimurium [s aureus . The anthimurium . Later, himurium . TherefoZS and analyze the differential metabolic pathways related to antimicrobial resistance. Glutathione metabolism and glutamic acid, aspartate, and glutamic acid metabolism were most significantly affected by the KEGG pathway. At the same time, the abundance of alanine, citric acid, and oxalic acid also decreased significantly. Our study showed that the metabolite abundance of Z173-S and Z173-RZS are very different, and the TCA cycle pathway may be related to the ZSM antimicrobial in Z173-RZS. These results were further verified by the fluctuation of the activity of three enzymes, i.e., SDH, PDH, and α-KGDH, in the TCA cycle and their corresponding gene transcription levels. The TCA cycle has been reported to be associated with bacterial antimicrobial resistance in previous work. For example, promoting the TCA cycle can increase the resistance to chloramphenicol in Edwardsiella tarda [Staphylococcus aureus with high drug resistance [Firstly, we used GC-MS-based metabolomic analysis to identify the intracellular metabolites in Z173-S and Z173-Rla tarda . Inhibitsistance .ZS. Our work showed that with the inhibition of the TCA cycle, the ZSM-controlling effect was also reduced. Inhibition of the TCA cycle is associated with the acquisition of antimicrobial resistance by Vibrio alginolyticus, Vibrio cholerae, and Pseudomonas aeruginosa [Secondly, the TCA cycle was inhibited by adding furfural, an inhibitor of pyruvate dehydrogenase, to test the relationship between the TCA cycle and the drug resistance in the ZSM-resistant strain, Z173-Rruginosa ,32,33. TSalmonella to apramycin [Edwardsiella tarda to kanamycin [ZS. Therefore, our work provides an applicable solution to controlling the ZSM-antimicrobial-resistant Xoo. Alanine could be used as a candidate in combination with ZSM, or as a synthetic precursor in developing new antimicrobials, for ZSM controlling in application. This study provides an important reference for the practical application of drugs against bacterial resistance and the development of new drugs. However, further experiments are needed to explore the specific mechanism of how the alanine functions in the TCA-associated ZSM-mediated controlling efficiency in Z173-RZS.Recent studies have shown that some key metabolites play a regulatory role in antimicrobial-related bacterial control. For example, exogenous fructose and maltose alter the antimicrobial sensitivity of Lan-negative bacteria through metabolic regulation . Exogenopramycin . On the anamycin . In the ZS with resistance to ZSM was isolated. These two bacteria were both grown at 30 °C for incubation.The antimicrobial-sensitive strain of Xoo (Z173-S) was provided by our laboratory. Through the subculture of Z173-S in PSA medium  containing 1/2 MIC of ZSM, the strain Z173-Rv/v) seed liquid was added in 24-deep-well plates with 2 mL of PSA liquid medium at 600 rpm and 30 °C shake cultivation. The biomass was valued at different time points  as the OD600 using the Multiskan Sky microplate reader. Three biological replicates were set for each time point.Z173-S initial cells from frozen stock were rapidly grown in PSA agar plates in a 30 °C incubator for static incubation for 2.5 d. A single colony was seeded in 3 mL of PSA broth medium at 200 rpm and 30 °C in a shaker overnight, and 1% . All experiments were carried out with biological triplicates.The single clones were cultured overnight at 30 °C with rigorous shaking of the tube containing 3 mL of PSA medium, followed by transfer to 24-deep-well plates equipped with 2 mL of PSA broth medium with 2-fold serially diluted ZSM at 0, 30, 60, 120, 240, 480, 960, and 1920 μg/mL at 600 rpm, 30 °C for 18 h. The value of ODZS were seeded into 50 mL of PSA liquid medium at 30 °C, 200 rpm to OD6001.0. The Xoo thallus were collected and centrifuged at 4 °C and 10,000 rpm for 5 min, and the supernatant was discarded and washed three times with 0.9% saline. After that, 10 mL of saline and 20 mL of frozen methanol were added immediately into the sample and set for at least 1 h to terminate the metabolic process of the cells. Bacterial cells collected were resuspended with 500 μL of frozen methanol with 10 μL of ribitol at 0.2 mg/mL as an internal reference. Cells were then sonicated at power 120 W for 5 min and centrifuged at 4 °C at 12,000× g for 10 min, and 500 μL of supernatant was dried with a nitrogen blower. Samples were then silanized and derivatized with 80 μL (20 mg/mL) of methoxyamine hydrochloride and 80 μL of N-methyl-N-trimethylmethilane trifluoroacetamide  and incubated at 37 °C, 200 rpm for 30 min, respectively. The samples were centrifuged at 4 °C at 12,000× g for 5 min and then analyzed with a GC-MS system. Each sample had five biological repeats.The sample preparation procedure was previously described . In briehttps://webbook.nist.gov/chemistry/ (accessed on 9 August 2022). For enrichment pathway analysis, the MetaboboAnalyst online website ) was used to determine the differential metabolic pathways involving the metabolites. Excel 2013 was used for data filtering and merge operations. The significant differences between metabolites in the control and experimental groups were compared using the Mann-Whitney U Test (p < 0.05) with SPSS 19.0 .GC-MS detection was carried out using the two-stage technique as described previously . BrieflyZS were cultured overnight and collected by centrifugation for 5 min at 8000 rpm, and the original nutrients were washed away with 0.9% normal saline of 30 mL. In the M9 minimum medium with 10.0 mM sodium acetate, 2.0 mM magnesium sulfate, and 0.1 mM calcium chloride, the samples were resuspended to 0.2 of OD600. In order to determine the effect of exogenous additions on Z173-RZS cells, the concentrations of alanine were set as 0, 0.01, 0.02, and 0.04 mol/L, and the concentration gradient of furfural was set to 0, 0.0425, 0.085, and 0.17 μL/mL. After incubation at 30 °C and 200 rpm for 6 h, the survival rate of Z173-RZS incubated at 30 °C for 36–48 h was calculated by continuous gradient dilution, and the CFU was counted. The survival rate was determined by dividing the CFU obtained from a treated sample by the CFU obtained from the control.The bacterial cells of Z173-R600, the 2 mL of liquid was collected and transferred to a new enzyme-free centrifuge tube. In the next step, the total RNA was extracted according to the total RNA extraction kit, and its concentration and purity were determined by full-wavelength enzyme labeling μDropTablet  then reverse transcribed into cDNA by one-step synthesis . The QRT-PCR reaction was carried out on a 96-well plate of the fluorescence quantitative PCR instrument . The primers are listed in −ΔΔCt method. At least three biological replicates were carried out.Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of enzymes related to the TCA cycle according to the method described above . In shorg for 10 min at 4 °C. The dehydrogenase activity was assayed in triplicate.The activities of three key enzymes—succinate dehydrogenase (SDH), pyruvate dehydrogenase (PDH), and α-ketoglutaric dehydrogenase (α-KGDH)—were carried out according to the instructions of the commercial enzyme activity detection kits , which were calculated by the protein concentration determined by BCA quantitative determination kit . The incubation of Xoo bacteria was performed as mentioned above. In short, exogenous additions  were added separately to the PSA medium overnight to centrifugate at 4 °C, 8000 rpm for 5 min. Before determination, the bacterial cells were fragmented with an ultrasonic crusher . The supernatants were collected by centrifugation at 11,000× ZS and a positive effect of alanine in reversing antimicrobial resistance was identified. In addition, we proposed an applicable candidate, alanine, to be used to enhance the antimicrobial-mediated Z173-RZS controlling efficiency. In subsequent studies, we will consider reprogramming functional metabolic pathways by metabolic regulators or combining other omics, including transcriptomics, whole genome sequencing, and proteomics, to explore the resistance mechanism of drug-resistant Xoo through metabolism, transcription, gene, and protein levels.In summary, we demonstrated that the metabolite abundance of susceptible and resistant bacteria is significantly different and that specific biomarkers are associated with ZSM resistance. In contrast to our previous studies, the mechanism of ZSM resistance in Xoo was further revealed. Our work confirmed that down-regulation of the TCA cycle is a crucial metabolic feature of Z173-R